presents (Basic) Laboratory Techniques (in Molecular Biology) A Montagud E Navarro P Fernández de Córdoba JF Urchueguía DNA cloning reading and writing DNA cut and paste DNA DNA sequencing bacterial transformation DNA synthesis transfection molecular hybridization chromosome integration Southern blot cellular screening Northern blot cellular culture Western blot extraction of DNA rewriting DNA : mutations Polymerase Chain Reaction random mutagenesis DNA polymerase DNA dependent point mutation PCR dynamics chromosome mutation PCR types arrays Gel electrophoresis DNA array protein array Laboratory Techniques DNA cloning DNA cloning overview Laboratory Techniques cut and paste DNA join two DNA molecules insert : usually smaller vector : has origin of replication Figure 8‐30. The insertion of a DNA fragment into a bacterial plasmid with the enzyme DNA ligase. (Alberts et al, 2002) Laboratory Techniques cut and paste DNA : vectors bacteria : plasmids restriction sites resistance gene origin of replication Figure 3.22. (Cooper, 2000) eukaryote : viruses restriction sites virus genes terminal repeats Laboratory Techniques cut and paste DNA : restriction restriction nucleases enzymes cut DNA from unspecific (exonucleases) to highly specific (type II endonucleases) leaves blunt or sticky ends rotational symmetry !! Figure 8‐22. Laboratory Techniques(Alberts et al, 2002) Figure 8‐21. (Alberts et al, 2002) cut and paste DNA : restriction why sticky ends ? Figure 8‐21. (Alberts et al, 2002) Figure 7‐7. Ligation of restriction fragments with complementary sticky ends. (Lodish et alLaboratory, 2000) Techniques cut and paste DNA : ligation DNA ligase Figure 5‐14. The reaction catalyzed by DNA ligase. (Alberts et al, 2002) Figure 7‐7. Ligation of restriction fragments with complementary sticky ends. (LodishLaboratoryet al, 2000) Techniques bacterial transformation make cell membrane permeable introduce plasmid DNA into bacteria many different protocols Figure 7‐16. Bacterium undergoing transformation. (Grifftihs et al, 2000) Laboratory Techniques transfection use of viruses to get DNA into cells in eukaryote and bacteria (transduction) Figure 7‐26. The mechanism of generalized transduction. (Griffiths et al, 2000) Laboratory Techniques chromosome integration how to make stable mutants adds a gene to a chromosome homologous recombination target DNA possible in bacteria usually necessary in eukaryote Figure 5‐75. Transpositional site‐specific recombination by a retrovirus or a retroviral‐like Laboratoryretrotransposon. Techniques (Alberts et al, 2002) cellular screening Figure 12‐6. Two plasmids designed as vectors for DNA cloning, showing general structure and restriction sites. (Griffiths et al, 2000) Laboratory Techniques cellular culture get lots of cells solid or liquid media time varies E. coli : overnight human cells : days adapted from AlbertsLaboratoryet alTechniques, 2002 extraction of DNA take only DNA from the whole cell extract brake cell membrane precipitate proteins purify DNA from proteins bound, etc Laboratory Techniques extraction of plasmids plasmids small circular independent DNA molecules extract only plasmids take advantage smaller, more compact a.k.a. maxi/midi/mini‐ prep Figure 12‐2. (GriffithsLaboratory et al Techniques, 2000) DNA cloning overview, again restriction + ligation plasmid extraction transformation cell cellular culture screening Laboratory Techniques movie : DNA cloning Ch7anim1. Lodish et al, 2000 Laboratory Techniques Polymerase Chain Reaction : copying DNA DNA polymerase DNA dependent copies DNA into DNA → DNA replication adds dNTP to a 3’OH end of an existing strand Figure 5‐4. DNA synthesis catalyzed by DNA polymerase. (Alberts et al, 2002) Laboratory Techniques Polymerase Chain Reaction double‐stranded DNA primers heat tolerant DNA polymerase (Taq pol) dNTPs 95ºC 55ºC 72ºC Figure 8‐39. Amplification of DNA using the PCR technique (Alberts et al, 2002) Laboratory Techniques PCR dynamics 95ºC 72ºC 55ºC Laboratory Techniques Polymerase Chain Reaction Figure 8‐39. Amplification of DNA using the PCR technique. (AlbertsLaboratory Techniqueset al, 2002) PCR amplifies DNA copies Laboratory Techniques movie : PCR Ch7anim4. Lodish et al, 2000 Laboratory Techniques PCR types Q‐PCR Allele‐specific PCR Assembly PCR Colony PCR Inverse PCR Ligation‐mediated PCR Nested PCR and many more... Laboratory Techniques Gel electrophoresis : separating molecules per length Gel electrophoresis nucleic acids in an agarose gel electric current through the gel nucleic acids DNA or RNA migrate to the + pole P skeleton has – charge separated per length Figure 7‐22. Separation of DNA fragments of different lengths by gel electrophoresis. dyed (EtBr, SYBR green) Laboratory Techniques (Lodish et al, 2000) Gel electrophoresis usually the length is – inferred using a known sample proteins SDS‐PAGE gel can be 2D: pI and weight + an EtBr gel electrophoresis Laboratory Techniques Figure 8‐17. (Alberts et al, 2002) reading and writing DNA DNA sequencing allows to know what is the exact sequence of A, T, C, G of a DNA molecule Sanger method (1977) based on PCR reaction & gel electrophoresis ddNTPs radioactively labelled Laboratory Techniques DNA sequencing Figure 8‐36. The enzymatic —or dideoxy— method of sequencing DNA. (Alberts et al, 2002) Laboratory Techniques movie : Sanger sequencing Ch7anim3. Lodish et al, 2000 Laboratory Techniques DNA sequencing : present & future fluorescence labels capillar electrophoresis polonies nanopores pyrosequencing Laboratory Techniques DNA synthesis commercial synthesis current price : from 0,80 €/bp production time : from 2 weeks from Carlson, 2003 Laboratory Techniques molecular hybridization molecular hybridization nucleic acids specifically hybridize to nucleic acids using labelled n.a., specific detection is possible Figure 5‐57. DNA hybridization. (Alberts et al, 2002) Figure 7‐17. Membrane‐hybridization assay for detecting nucleic acids. Laboratory Techniques (Lodish et al, 2000) molecular hybridization in combination with gel electrophoresis, detection boasts its potential Figure 8‐27. Detection of specific RNA or DNA molecules by gel‐transfer hybridization. (AlbertsLaboratoryet al ,Techniques 2002) molecular hybridization Southern blot (DNA) Northern blot (RNA) DNA extraction RNA extraction restriction denaturation gel electrophoresis gel electrophoresis denaturation filter transfer filter transfer labelled probe hybridisation labelled probe hybridisation DNA DNA or RNA detection detection Western blot (protein) polyacrilamide gel separation probe reaction filter transfer antibody Laboratory Techniquesdetection Southern, Northern, Western Figure 10‐20. Comparison of Southern, Northern, and Western analyses of Gene X. (Griffiths et al, 2000) Laboratory Techniques rewriting DNA : mutations mutations variations on a given DNA molecule basis of variability → evolution small‐scale types silent mutation : a.a. is not afected missense mutation : different a.a. nonsense mutation : a.a. → stop Figure 8‐4. Different types of mutations. (Lodish et al, 2000) Laboratory Techniques mutations large‐scale types : chromosomes inversion : changes order insertion : adds genes deletion translocation : moves genes Figure 8‐4. Different types of mutations. (Lodish et al, 2000) Laboratory Techniques random mutagenesis use chemicals, UV or error‐prone DNA replication fine screening needed ! Figure 16‐2. Mismatched bases. Figure 5‐49. How chemical modifications of (Griffiths et al, 2000) nucleotidesLaboratory Techniques produce mutations. (Alberts et al, 2002) point mutation point mutation on a given site PCR is widely used for this goal site‐directed megaprimers in vitro overlap‐ extension Figure 8‐69. The use of a synthetic oligonucleotide to modify the protein‐coding region of a gene by Laboratory Techniques site‐directed mutagenesis. (Alberts et al, 2002) movie : PCR mutagenesis Ch8anim1. Lodish et al, 2000 Laboratory Techniques chromosome mutation mutate homologous chromosomes recombination knock out target DNA replace insertion : add a gene deletion Figure 8‐64. Gene replacement, gene knockout, and gene addition. (Alberts et al, 2002) Laboratory Techniques chromosome mutation Figure 8‐73. Making collections of mutant organisms. (Alberts et al, 2002) Laboratory Techniques arrays DNA array probe DNA molecules of variable lenght on a solid support in a regular and fixed distribution sample labelled DNA or RNA that will bind to the probes Figure 14‐27. (Griffiths et al, 2000) take advantage of nucleic acid’s specific hybridization Laboratory Techniques expression array probe usually organism’s ORFs ordered (oligo chip) sample usually labelled mRNA or retrotranscribed mRNA (cDNA) Laboratory Techniques expression array monitor mRNA quantities of the whole genome compare two states inputs outputs sample 1 > sample 2 sample 1 sample 1 < sample 2 sample 2 sample 1 = sample 2 Figure 8‐62. Using DNA microarrays to monitor the expression of Laboratory Techniques thousands of genes simultaneously. (Alberts et al, 2002) expression array : breast cancer 1753 84 genes number of experiments below above median median median Perou et al (2000) Laboratory Techniques expression array : breast cancer Perou et al (2000) Laboratory Techniques protein array probe protein molecules on a solid support in a regular and fixed distribution sample substrates that will